The therapeutic utility of botulinum neurotoxin type A (BoNT/A) has grown considerably over the past several decades and Allergan's product, onabotulinumtoxinA, is now approved globally for 11 major therapeutic and cosmetic indications, including treatment for various neuromuscular disorders (Ramirez-Castaneda, J. and Jankovic, J., (2014). “Long-term efficacy, safety, and side effect profile of botulinum toxin in dystonia: a 20-year follow-up.” Toxicon 90, 344-348.; Yablon, S. A., Brin, M. F., VanDenburgh, A. M., Zhou, J., Garabedian-Ruffalo, S. M., Abu-Shakra, S., and Beddingfield, F. C., III (2011). “Dose response with onabotulinumtoxinA for post-stroke spasticity: a pooled data analysis.”Mov Disord. 26, 209-215.), smooth muscle and autonomic dysfunctions (Ellsworth, P. and Travis, M., (2014). “Onabotulinum toxin A: a therapeutic option for refractory neurogenic detrusor overactivity and idiopathic overactive bladder.” Urol. Nurs. 34, 165-171.; Grunfeld, A., Murray, C. A., and Solish, N., (2009). “Botulinum toxin for hyperhidrosis: a review.” Am. J. Clin. Dermatol. 10, 87-102.) and for nociceptive pain syndromes (Aoki, K. R. and Francis, J., (2011). “Updates on the antinociceptive mechanism hypothesis of botulinum toxin A.” Parkinsonism. Relat Disord. 17 Suppl 1, S28-S33.; Burstein, R., Zhang, X., Levy, D., Aoki, K. R., and Brin, M. F., (2014). “Selective inhibition of meningeal nociceptors by botulinum neurotoxin type A: therapeutic implications for migraine and other pains.” Cephalalgia 34, 853-869.).
While the general mechanism of action (MoA) for BoNT/A at the presynaptic nerve terminal is well established (Montal, M., (2010). “Botulinum neurotoxin: a marvel of protein design.” Annu. Rev. Biochem. 79, 591-617.), there are still many unanswered questions regarding the intracellular trafficking patterns and general ‘life-cycle’ of the toxin. Resolving these questions partly depends on the ability to precisely detect the toxin's location, distribution, and movement within a cell. Direct detection of BoNT/A using antibodies is difficult due to its high potency and therefore, extremely low concentration within neurons. An alternative approach for detecting the presence of BoNT/A has been to track its enzymatic activity via immuno-staining for the cleaved SNAP25 product (SNAP25197). Both commercial and proprietary antibodies have been used to trace the expression of full-length SNAP25 (SNAP25206) or BoNT/A-cleaved SNAP25 (SNAP25197). However, the terminal epitope of SNAP25197 that is generated following BoNT/A cleavage is difficult to specifically target with an antibody without also recognizing the intact SNAP25 protein (Mort, J. S. and Buttle, D. J., (1999). “The use of cleavage site specific antibodies to delineate protein processing and breakdown pathways.”Mol. Pathol. 52, 11-18.). Consequently, immuno-staining results have been misleading, with some antibodies being assay-dependent while other antibodies are tissue-specific. There is therefore a need for a very selective antibody against BoNT/A-cleaved SNAP25 that can identify SNAP25197 in any tissue-type and in multiple assays following exposure to BoNT/A.
The present disclosure addresses these issues by providing methods and compositions for detecting botulinum toxin cleaved SNAP25, including BoNT/A cleaved SNAP25 and any other BoNT/A-related compounds that cleave SNAP25 at position ‘197’, in various different assays, such as but not limited to immunohistochemistry using highly specific recombinant monoclonal antibodies (rMAb) against SNAP25197. These antibodies can be used to detect SNAP25197 in a variety of tissues from different species, such as but not limited to human. These antibodies can also be used as tools to diagnose activity and efficacy in tissues from humans that have been treated with neurotoxin, such as but not limited to onabotulinumtoxinA.